Tyrosinase in the Experiment - Lab Report Example

Tyrosinase Tyrosinase involves an oxidoreductase containing copper and catalyses the the monophenols orthohydroxy-lation and the catechols of aerobic oxidation. In this experiment the activity of the enzyme was assessed by monitoring the oxidation of dopa (3, 4-dihydroxyphenyl alanine) to dopachrome which is red in colour. The kinetic parameter such as Km and V were evaluated using direct linear plots. The manner in which thiourea inhibits tyrosinase were also be explored. In this study one steroisomer (L-dopa) was tested as substrate. Introduction. In biology enzymes are classified as catalysts. Absence of enzymes in any cell means than many biochemical reactions would not occur at the expected rate. The biochemical and the physiochemical reactions of enzymes have been advanced from the early 1800s as a result of the interest in the enzyme dynamism, selectivity, catalytic power and their function in the cell. This study will explore one dynamic trait of enzymes which is the enzyme kinetic description. In many cases, the reactions that are catalysed by enzymes follow an ES complex format shown in equation 1. Equation 1 E9E +S ES E + P, where E is the enzyme, P is the product, and S is the substrate (Kumar, C. 2011). For an enzyme that is specific, one substrate molecules may bind in the required way to give out a function of the EES complex. On the other hand, the substrate needs to have a shape, size and polarity that is compatible with the active enzyme site (Witkop, C. 2009). Other enzymes may catalyse the many different molecules transformation whenever there exist a common category of a chemical linkage of the given substrate. Some have specificity hence can form ES complexes that are reactive having one structure of a molecule. Enzyme kinetic properties The first velocity of reaction (V0) of a reaction catalysed by an enzyme is variable in accordance to the concentration of the substrate (s). The Michaelis Menten equation was establish to account to the kinetic properties of the different enzymes. This equation is given as; V0 = JTK. Material and methods The reagents were dissolved in the phosphate buffer. These reagents included a combination of enzyme, phosphate buffer, inhibitor of the specific experiment and the substrate. The required phosphate buffer volume was pipetted together with the substrate and the inhibitor into the cuvette. The solution was well mixed and the coloximeter on the cuvette zeroed. The cuvette was removed from the colorimeter. The needed volume of the enzyme was quickly added and mixed through covering the cuvette using paraffin. The stop clock was then started. The cuvette was placed in the colorimeter and the initial reading taken at fifteen seconds. The recording process continued at an interval of fifteen until two minutes. The final cuvette volume of the experiment was 3.0 cm cubed. Part A To determine a suitable tyrosinase concentration for the analysis of the kinetics, the experimental set up was set up and ran as displayed in table 1. The volume of the enzyme was varied whereas the substrate was kept constant. Table 1 Reagent Cuvette 1 2 3 4 L-DOPA (S) 1.5 1.5 1.5 1.5 Tyrosinase (E) 0.05 0.1 0.2 0.4 Phosphate buffer 1.45 1.4 1.3 1.1 Inhibitor Concentration [I] 1 1 1 1 Part B To determine the Michaels constant, Km for L-DOPA, the experimental set up was run at a constant while varying the substrate. The AAm/min for the enzyme volume was estimated. The tyrosinase volume was selected giving a AAA/min of about 0.2 - 0.25 and using the volumes of LOPA displayed in table 2. The volume of the buffer was calculated to obtain a volume of 3.0 cm cubed. Table 2 Reagent Cuvette 1 2 3 4 5 6 L-DOPA (S) 1.5 0.8 0.4 0.2 0.1 0.0 Tyrosinase (E) 0.4 0.4 0.4 0.4 0.4 0.4 Phosphate buffer 1.1 1.8 2.2 2.4 2.5 2.6 Substrate concentration (S) 1 0.533 0.26673 0.1333 0.06667 0 1/S 0.005 0.00938 0.01876 0.0375 0.07499 0 Part c To determine the effects of inhibiting benzoic acid and thiourea, each pair investigated the activity of the inhibitors. The assays were run at an enzyme that is constant and a fixed concentration of the inhibitor. The enzyme and L-DOPA volumes were used as in the prior sections. The required L-DOPA volume was mixed together with the buffer and inhibitor as shown in table 3. Each reaction was started individually by adding the selected enzyme volumes that were used earlier. Table 3. Reagent Cuvette 1 2 3 4 5 6 L-DOPA (S) 1.5 0.8 0.4 0.2 0.1 0.0 Tyrosinase (E) 0.4 0.4 0.4 0.4 0.4 0.4 Inhibitor (Thiourea) 0.5 0.5 0.5 0.5 0.5 0.5 Phosphate buffer 0.6 1.3 1.7 1.9 2.0 2.1 Substrate Concentration [S] 1 0.533 0.26673 0.1333 0.06667 0 1/S Results The absorbance at an interval of fifteen minutes for part a, part b, and part c was recorded as shown in tables 4, 5, and 6. Table 4: Part a TIME 15 30 45 60 75 90 105 120 TUBE 1 0.02 0.025 0.03 0.04 0.05 0.06 0.07 0.08 2 0.04 0.06 0.08 0.09 0.11 0.13 0.14 0.155 3 0.04 0.08 0.11 0.15 0.18 0.21 0.23 0.25 4 0.09 0.16 0.23 0.29 0.36 0.41 0.46 0.51 Graph 1 Table 5: Part b TIME 15 30 45 60 75 90 105 120 TUBE 1 0.09 0.16 0.24 0.30 0.36 0.42 0.47 0.52 2 0.07 0.13 0.18 0.25 0.30 0.35 0.40 0.45 3 0.05 0.10 0.14 0.18 0.23 0.27 0.31 0.35 4 0.005 0.09 0.13 0.16 0.20 0.24 0.27 0.31 5 0.04 0.07 0.11 0.14 0.17 0.20 0.23 0.26 6 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Graph 2 Table 6: Part c TIME 15 30 45 60 75 90 105 120 TUBE 1 0.04 0.08 0.11 0.15 0.18 0.21 0.25 0.28 2 0.04 0.07 0.11 0.14 0.17 0.20 0.23 0.26 3 0.04 0.08 0.12 0.15 0.18 0.21 0.24 0.27 4 0.04 0.07 0.11 0.14 0.16 0.18 0.21 0.23 5 0.04 0.07 0.09 0.12 0.14 0.16 0.18 0.20 6 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Graph 3 From the graph the change in absorbance per minute for part a, part b and part c was determined as shown in table 7 table 8 and 9. Table 7: part a. Tube Absorbance per min 1/V 1 0.0006 1666.7 2 0.0011 909.1 3 0.002 500 4 0.004 250 Table 8: Part b Tube Absorbance per min 1/V 1/S 1 0.0041 243.9 0.005 2 0.0036 277.8 0.00938 3 0.0028 357.1 0.01876 4 0.0027 370.3 0.07499 5 0.0021 476.1 0 6 0 0 From the graph, the gradient= 8262.7, and y-intercept= 201.66 Table 9: Part c Tube Absorbance per min 1/V 1/S 1 0.0023 434 0.005 2 0.0022 454.5 0.01878 3 0.0021 476.1 0.07499 4 0.0018 555.6 5 0.0015 666.7 6 0 0 From the graph, the gradient= 1618, while the y-intercept= 436.31. Discussion This is clearly a linewaver-Burk Plot, which illustrates the kinetic data. The two graphs (b and c) represents Michaelis-Menten equation. The produce a straight line defined by equation y = mx + c. The y-intercept is equivalent to 1/Vmax and the x-intercept represents −1/KM. alternatively, basing on the equation 2 below, y-intercept represents 1/Vmax, and the slope of the graph represents Km/1/Vmax. This implies that in b 1/Vmax = 201.66, and Vmax= 0.004956, and Km= 8262.7/201.66= 40.973. Using similar analogy in C the value for 1/Vmax = 436.31, and Vmax= 0.00229, and Km= 1618/436.31= 3.708. …………………………………………..equation 2 The study found out that there was some changes in the relationship between substrate concentration and the initial rates. When the substrate concentration is low, the initial rate will varies linearly (V0= Ki(S). A relationship of this kind is referred to as the first order kinetics (Kumar, C. 2011). Whenever the concentration of the substrate is high, the initial draws towards a constant reaction (V0=K). This is not depended of the increasing concentration of the substrate. This is displayed in graph 1, 2 and 3. The rate at high concentration of the substrate is referred to as Vmax. This means that at high concentration of substrate V= K(release)= Vmax. On the other hand, at a low concentration of the substrate, the primary rate contribution is K1/K2, which is the ES complex rate formation and is depended on the concentration of the substrate. This is a kinetic constant referred to as the Michaelis constant. Michaeli constant involves a constant of equilibrium for ES complex formation, and approximately equal to the constant for the substrate (Chang, T. 2009). References Kumar, C., 2011. The interaction between sesamo; and tyrosinase. Biochimie 93 (3): 562–9.  Kumar, C., 2011. The human gene tyrosinase, mapped to chromosome 11 defining second homological reasons with mouse chromosomes. Genomic 3 (1): 17–24.  Witkop, C., 2009. The Albinism: Disease hematologic-storage, susceptible to skin cancer, and the optic neuronal defects that are shared in all types of ocular and oculocutaneous albinism. Ala J Med Science 16 (4): 327–30.  Chang, T., 2009. The Tyrosinase inhibitors. International J Mol Sci 10 (6): 2440–75.  Read More